- Title
- Cleavage of the low-density lipoprotein-receptor-related protein (lrp) by matrix metalloproteinases (mmps) in a neural cell model: identification of multiple soluble lrp (slrp) cleavage products
- Creator
- Fitzsimmons, Chantel
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The low-density lipoprotein receptor-related protein (LRP) is a large transmembrane scavenger and signalling receptor. Binding over 50 ligands in the extracellular environment LRP has a wide range of physiological and pathological functions. For example, in the brain, LRP ligands include the Alzheimer’s disease amyloid beta peptide (Ab) and apolipoprotein E (APOE). Cellular LRP can be cleaved by proteolytic enzymes to generate soluble fragments (sLRP). Some sLRP fragments retain one or more ligand-binding domains and may exert antagonistic effects on cellular LRP. Previous studies using broad spectrum matrix metalloproteinase (MMP) inhibitors suggest one or more MMPs may be implicated in sLRP generation. The MMPs are a group of zinc-dependent endopeptidases that are best characterised for their ability to degrade extracellular matrix (ECM) proteins and remodel existing cell-matrix boundaries. The broad hypothesis underlying this project is that MMPs can contribute to the generation of sLRP species in a cellular system relevant to the CNS. The SH-SY5Y cell line, derived from a human neuroblastoma, was established as a useful cell model. The cell line was found to endogenously release several putative sLRP a-chain (~500 kDa) and b-chain (~275 kDa, ~150-100kDa, ~85 kDa, ~75 kDa, ~65 kDa and ~55 kDa) species in vitro. While developing this model under serum-free conditions, substantial quantities of sLRP immunoreactivity were detected in the B27 supplement that was not listed among the components. The unrecognised presence of sLRP, and possibly other undefined serum proteins, has the potential to introduce experimental artefacts and interfere with experimental results and interpretation in cell culture studies involving LRP or any of its ligands. Neurobasal medium supplemented with N2 was identified as an alternative serum-free medium suitable for investigating sLRP production in vitro. Treatment with synthetic MMP inhibitors reduced the release of soluble LRP species into the culture medium. It was found that sLRP is generated primarily by MMP-2 in this system and possibly also by MMP-9 dependent activity. The data provided evidence that LRP contains at least two MMP cleavage sites: at least one located within the a-chain and one in the b-chain. N-terminal sequencing using Edman degradation and MALDI-ToF did not generate sequence data due to N-terminal blockage. However all the soluble species, with the exception of the ~75 kDa and ~65 kDa species, were no longer detectable after RNA interference was used to silence LRP gene function. This confirms that these species are soluble forms of cellular LRP. This also shows that the ~75 kDa and ~65 kDa species are not in fact derived from LRP. RNA interference was also utilised as an alternative approach to reduce the levels of specific MMP transcripts within the cell model. In accordance with the MMP inhibitor studies it was found that sLRP production is MMP-2 and MMP-9 mediated. As MT1-MMP is required for the activation of pro-MMP and potentially also pro-MMP-9, the effect of down-regulating MT1-MMP gene function was also investigated. Production of the major ~500 kDa sLRP-a species and the major ~85 kDa and ~55 kDa sLRP-b species was almost completely abolished, proving evidence that MT-MMP has important roles in sLRP generation in this system. In summary, the findings presented within this thesis provide evidence for the first time that several sLRP species are endogenously produced in cells relevant to the CNS. Furthermore, data is also presented that identifies MT1-MMP, as well as MMP-2 and MMP-9, as the key matrix metalloproteinase able to affect sLRP production in a neural cell system. Understanding the mechanisms by which LRP is processed may help develop new treatment strategies for Alzheimer’s disease or related neurodegenerative disorders.
- Subject
- soluble LRP; matrix metalloproteinase
- Identifier
- http://hdl.handle.net/1959.13/935783
- Identifier
- uon:12136
- Rights
- Copyright 2012 Chantel Fitzsimmons
- Language
- eng
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| View Details Download | ATTACHMENT02 | Thesis | 2 MB | Adobe Acrobat PDF | View Details Download |